Basic Principles of Analytical Ultracentrifugation

Product Description

Analytical ultracentrifugation (AUC) consists of the application of a high gravitational field to a solution of particles and the real-time detection of the evolving spatial concentration gradients. When applied to macromolecules or nanoscopic particles, ultracentrifugation data can provide rich information on their shape, solvation, composition and size-distribution, as well as allow for a detailed view of their reversible single- or multi-component interactions over a wide range of affinities. After almost a century of methodological development, stimulated by ever-changing emphasis in applications, as well as substantial instrumental and computational advancements, a wealth of theoretical and experimental knowledge of sedimentation has been accumulated. Unfortunately, no systematic textbook or comprehensive monograph on AUC has been published since the initial work by Svedberg and Pedersen 1940, and the seminal detailed methodological summary by Schachman, 1959. This has rendered AUC a discipline that is hard to master without direct access to experienced laboratories where it is routinely practiced, and hampers this powerful technique from once again becoming a mainstream tool in the molecular sciences.

The goal of the present book is to provide a description of the basic principles in theory and practice, sufficiently comprehensive for the reader to confidently practice AUC, and to be aware of its full potential and possible pitfalls. The book aims to help the reader gain a solid understanding of the basic concepts, and to facilitate further reading of the referenced detailed topics, with appreciation for their historic and current relevance. The emphasis is experimental, and more detailed descriptions of the theoretical frameworks and data analysis strategies are planned in forthcoming volumes. Although we always strived to provide the most important and historically accurate references, we recognize that ambiguities exist, and apologize for any perceived omissions or limitations in our knowledge.

The first chapter introduces the basic principles and technical setup of an analytical ultracentrifugation experiment, together with a brief description of the optical systems used for detection. The ultracentrifugation experiment is subsequently explored in Chapter 2 from a macromolecular standpoint to arrive at a detailed physical picture of the sedimentation process, from which to derive the relevant macromolecular parameters. Next, we recapitulate important practical aspects for conducting an experiment, including sample preparation (Chapter 3), details on data acquisition and data structure (Chapter 4), and the practical execution of the centrifugal experiment (Chapter 5). Instrument calibration and quality control experiments are outlined in Chapter 6. Tables of often useful data for AUC, including the properties of common macromolecules and solvents, are assembled in the appendices.

Throughout, to enrich the utility of the book and illustrate the facility of practical application of ideas ranging from simple to advanced, specially marked textboxes highlight how the topic at hand corresponds to AUC-related functions in the widely used data analysis programs SEDFIT and SEDPHAT, which can be freely obtained from the website of the Dynamics of Macromolecular Assembly Section of the National Institute of Biomedical Imaging and Bioengineering at sedfitsedphat.nibib.nih.gov. Although the book is conceived as a standalone reference, it also provides a broader background to our workshops on AUC and related biophysical techniques at the National Institutes of Health.